Figure 5. Defects in neuron viability in Kir4.1 cKO mice.

(A) Kir4.1 cKO sympathetic neurons have smaller soma sizes compared to control neurons. Results are mean ± SEM from n = 3 control and 5 mutant mice, ***p<0.001, two-way ANOVA with Bonferroni’s correction. (B) Reduced soma size, represented as median values of soma areas (μm²) of Kir4.1 cKO sympathetic neurons compared to controls. Values are mean ± SEM from n = 3 control and 5 mutant animals, *p<0.05, t-test. (C) Decreased mTOR signaling based on p-S6 immunostaining in Kir4.1 cKO sympathetic ganglia. Scale bar: 100 μm. (D) TUNEL labeling shows increased apoptosis in Kir4.1 cKO SCG (outlined in red dashed line). Scale bar: 100 μm. (E) Quantification of apoptotic cells in control and mutant sympathetic ganglia from n = 3 mice per genotype. Data presented as mean ± SEM **p<0.01, t-test. (F, G) Decreased sympathetic neuron numbers in Kir4.1 cKO mice based on Nissl-staining and cell counts in sympathetic ganglia tissue sections. Results are mean ± SEM from n = 3 control and 4 mutant animals. *p<0.05, t-test. (H) Heart rate is unaffected by Kir4.1 deletion from satellite glia. Results are mean ± SEM from n = 5 control and 6 mutant animals, two-way ANOVA with Bonferroni’s correction. (I) Average heart rate over days 4–7 post-lead implantation. Results are mean ± SEM from n = 5 control and 6 mutant animals. n.s., not significant.

Figure 5—figure supplement 1. Additional morphology and functional analyses in Kir4.1 cKO mice.

(A) Reduced p-4E-BP1 levels as shown by immunostaining in Kir4.1 cKO sympathetic neurons compared to controls. Scale bar: 100 μm. (B) Increased neuronal apoptosis in Kir4.1 cKO ganglia as shown by TrkA immunostaining (magenta) and TUNEL labeling (green). Arrowheads indicate TUNEL⁺;TrkA⁺ sympathetic neurons. Scale bar: 30 µm. (C) Quantification of TUNEL⁺;TrkA⁺ neurons expressed as a % of total number of TrkA⁺ neurons. Data are presented as mean ± SEM from n = 4 control and 5 mutant mice, *p<0.05, t-test. (D) Glial apoptosis is unaltered with Kir4.1 loss, assessed by Sox10 immunostaining (green) and TUNEL labeling (red). Arrowheads indicate TUNEL⁺; Sox10⁺ cells. Scale bar 30 µm. (E) Quantification of TUNEL⁺;Sox10⁺ glial cells expressed as a % of the total number of Sox10⁺ satellite glial cells. Data are presented as mean ± SEM from n = 6 control and 4 mutant mice, n.s, not significant, t-test. (F) Tyrosine hydroxylase (TH) immunostaining shows normal sympathetic axon innervation in Kir4.1 cKO salivary glands. Scale bar: 100 µm. (G) Quantification of sympathetic innervation density in salivary glands. Data are mean ± SEM from n = 6 control and 5 mutant mice, n.s, not significant, t-test. (H, I) Basal pupil areas are unaffected by satellite glia-specific Kir4.1 deletion. Data are mean ± SEM from n = 10 control and 9 mutant animals, two-way ANOVA with Bonferroni’s correction. (J) Circulating norepinephrine (NE) levels are unaltered with Kir4.1 deletion from satellite glia. Data are mean ± SEM from n = 7 control and 12 mutant animals, n.s., not significant, t-test. (K) Pupil constriction in response to light, indicative of parasympathetic activity, is normal in Kir4.1 cKO mice. Data are mean ± SEM from n = 5 control and 6 mutant animals, two-way ANOVA with Bonferroni’s correction.